ABSTRACT A-Kinase Anchoring Proteins (AKAPs) organize second messenger responsive enzymes to direct the flow of information within cells. These proteins were initially discovered as protein kinase A (PKA) binding partners. However, it is now clear that the primary function of AKAPs is to integrate a variety of intracellular signals. This occurs by sequestering PKA with other kinases, small GTPases, and protein phosphatases within range of their substrates. The physiological significance of this mechanism has been validated in several contexts. This proposal exploits new discoveries about AKAP220 signaling complexes to establish if manipulation of this macromolecular assembly is of therapeutic value in the restoration of water homeostasis to manage aspects of autosomal dominant polycystic kidney disease. Human kidneys filter about 180 liters of fluid every day, yet a majority of the water is reabsorbed, as only 1.5 liters of urine is excreted. Urine is concentrated in the kidney-collecting duct where water is reabsorbed from luminal fluid through aquaporin-2 (AQP2) water pores. The hormone arginine vasopressin (AVP) increases water permeability by inducing PKA phosphorylation of Ser256 on AQP2 to stimulate translocation of the water pore from vesicles to apical membranes of collecting ducts. Not surprisingly, defects in aquaporin-2 trafficking have dire pathophysiological consequences. Polycystic kidney disease occurs when fluid filled cysts grow in the kidney collecting ducts. These cysts eventually replace much of the kidneys and lead to kidney failure. Autosomal dominant polycystic kidney disease is a leading cause of end-stage renal failure worldwide. At the molecular level, mutations in the cilia transmembrane proteins polycystin 1 (PC1) and polycystin 2 (PC2) promote defective osmoregulation through aquaporin-2. Aberrant cAMP signaling, altered cilia assembly and reduced Rho GTPase activity further contribute to the expansion of fluid filled cysts. Our preliminary findings implicate AKAP220-binding partners in each of these pathological responses. An experimental plan of three specific aims is proposed. Aim 1 will employ state-of-the-art analytical and proximity ligation approaches to define the enzyme composition, stoichiometry and subcellular location of AKAP220 complexes. Aim 2 will investigate 3D organoid cultures to establish if AKAP220-associated GTPase effector protein IQGAP1 sustains actin barrier formation through local modulation of RhoA. Aim 3 will employ drug delivery to CRISPR/Cas 9 gene-edited kidney-derived cells in a microfluidic ?Kidney-on-a-chip? device to determine if AKAP220-associated phosphatase 1 (PP1) impacts signaling events that underlie aquaporin-2 trafficking and cilia biogenesis.